Elevated stress response marks deeply quiescent reserve cells of gastric chief cells

Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4+ population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration.


H
ierarchically organized tissues such as hematopoietic tissues, skeletal muscle, neuronal tissues, and hair follicles harbor quiescent cells as a source for regeneration of damaged tissues if incumbent stem cells are damaged or lost due to external insults 1,2 .The quiescent reserved cells in these organs, which often exhibit stem cell-like characteristics, maintain resident stem cell populations and organ homeostasis.
The gastrointestinal tract, which constantly faces direct environmental exposure, is also equipped with reserve cells to repair its damage.The process of regeneration after injury has been extensively studied in the small intestine of the lower gastrointestinal tract.In the absemce of damage, Lgr5 + actively-cycling stem cells, which are present at the bottoms of intestinal crypts, differentiate into all lineages in the crypt structure.In response to severe tissue damage, slow-cycling cells that are located at the +4 position are converted into fast-cycling Lgr5 + stem cells 3 .These observations suggest that +4 cells serve as cellular reservoirs of stem cells [4][5][6] .Alternatively, the conversion of the +4 cells into stem cells could be interpreted as dedifferentiation of progenitor cells 7,8 .
In the upper gastrointestinal tract, tissue regeneration after injury has been mainly studied in the stomach.The stomach is divided into several anatomically distinct regions, including the antrum and corpus 9 .The gastric units in the antrum and corpus both have an isthmus region that is the source of bidirectional proliferation of stem cells 9 .As is the intestine, Lgr5 + cells reside at both the gland base and +4 position in the antrum, and the expression of Lgr5 or Aqp5 in the gland base marks cycling stem cell populations.
Notably, in the corpus region, chief cells are an additional source of reserve cells.Chief cells are slow cycling and function as a reserve for regeneration of the gastric unit after severe damage 10,11 .Of note, lineage tracing assays have indicated that several chief cell-expressed genes, including Lgr5, Troy, Mist1, Gpr30, GIF, and p57, behave as markers of damage-responsive reserve cells [12][13][14][15][16][17] .Although these reports have demonstrated the plasticity of chief cells, the molecular basis of this phenomenon remains unclear.
Interestingly, during the regeneration, chief cells can undergo dramatic reprogramming resulting in a type of metaplasia known as spasmolytic polypeptide-expressing metaplasia (SPEM) 10,[17][18][19][20] .This metaplastic response is called paligenosis 21 , and has recently attracted attention as it was proposed that gastric cancer arises from the resulting metaplasia 20 .The dynamic process of paligenosis involves several stages, and transient inhibition of mTORC1 and activation of the autophagic/lysosomal response are followed by induction of metaplastic genes, including Atf3 22 or Sox9 23 .
In this paper, we systematically searched for slow-cycling reserve cells in the epithelia of digestive organs.Previously, H2B-GFP label-retaining assays have been successfully applied to identify quiescent cells in diverse organs [24][25][26][27][28][29] .Therefore, to evaluate the presence of quiescent reserve cells in the gastrointestinal tract, we performed a label-retaining assay after temporary H2b-GFP expression.Remarkably, we detected GFP + epithelial cells for >2 months after labeling in the gastric corpus but not in other digestive tract organs.Expression analyses demonstrated that the GFP + subpopulation of chief cells specifically expressed Atf4.Furthermore, these GFP + cells started proliferation after tissue damage, indicating that the identified quiescent cells share phenotypic similarity with cells undergoing paligenosis upon tissue damage.

Results
Gastric epithelia specifically harbor label-retaining long-term quiescent cells.To investigate the presence of long-term quiescent cells in the gastrointestinal tract, we performed long-term labeling with an inducible chimeric mouse histone 2B-green fluorescent protein (H2b-GFP).To do this, H2b-GFP regulated by a tetracycline operator (TRE3G) was knocked in at the Rosa26 locus together with a third-generation tetracycline-inducible transactivator (Tet-on 3 G) 30 to generate Rosa26 iH2b-GFP mice (Fig. 1a and Supplementary Fig. 1a).In Rosa26 iH2b-GFP mice, the fusion protein was widely induced in the gastrointestinal epithelia after oral ad libitum administration of doxycycline.Rosa26 iH2b- GFP mice were subjected to H2b-GFP pulse-labeling for 4 weeks, which was followed by chase periods in the absence of doxycycline (1-9 weeks).Subsequently, the gastrointestinal tracts of the labeled mice were examined by immunostaining and flow cytometry (Fig. 1b and Supplementary Fig. 1b).
Flow cytometric analyses of the stomach, small intestine, and colon indicated that Epcam + epithelial cells in these organs were pulse-labeled with H2b-GFP with varying frequencies (stomach: 17.1%, small intestine: 53.0%, colon: 57.3%) (Fig. 1c, 0 weeks after doxycycline treatment).In the small intestine and colon, cell proliferation caused rapid loss of GFP signals in the GFP + population, and GFP + cells were nearly undetectable within 3 weeks.In contrast, in the stomach, 2.9% of GFP + cells (0.5% out of the initially labeled population (17.1%)) retained detectable GFP signals after 6 weeks (Fig. 1c).
To validate the unique retention of pulse-labeled H2b-GFP in the stomach epithelial subpopulation, we examined retained H2b-GFP expression in multiple gastrointestinal organs using immunostaining.Remarkably, GFP + cells were detected in the lower area of the gastric corpus even 9 weeks after the start of the chase periods (Fig. 1d).The GFP-labeled cells were E-Cadherin (Cdh1)-positive, indicating that these cells were of epithelial origin (Fig. 1d).In contrast, GFP-labeled cells were almost undetectable after 3 weeks in other gastrointestinal organs including the esophagus, forestomach, antrum, jejunum, and colon (Supplementary Fig. 1c), indicating that the presence of long-term quiescent cells in the stomach was unique among organs of the gastrointestinal tract.
Deeply quiescent cells in the stomach are localized in the gastric corpus.To identify the cell-types associated with the GFP + label-retaining corpus epithelial cells, we performed singlecell RNA-seq analyses of FACS-sorted Epcam + cells and Epcampositive GFP + cells (GFP + cells) (Supplementary Fig. 1b).Based upon gene expression profiles, the Epcam + cells were stratified into 10 subpopulations (Fig. 2a).When we combined the GFP + cells with the Epcam + cells (mixed at a ratio of ~4.5 to 1; hereafter called GFP-mixed cells), we could stratify the mixture into the same 10 subpopulations (Fig. 2a), which allowed us to evaluate the cell types of the GFP + cells in the following studies.
Chief cells were reported to harbor quiescent stem cells with distinct markers 33 .Indeed, Lgr5 and Troy were highly expressed in cluster 5, and Mist1 was highly expressed in both clusters 1 and 5.As expected, Bmi1 and Stmn1, markers of isthmus stem cells 11,36 , were not highly expressed in either cluster 1 or 5 (Fig. 2c).
We subsequently determined whether the GFP + cells could be classified into distinct subpopulations.The GFP + cells were preferentially enriched in cluster 1 (Fig. 2d).Coimmunostaining indicated that the GFP + cells near the basal layers did not express markers specific for isthmus cells (Mki67), Pit cells (Krt19), Tuft cells (Dclk1), or endocrine cells (Chga) (Supplementary Fig. 2b) but did express Pla2g1b, a chief cell marker (Fig. 2e, f).These data  indicate that the GFP + cells near the basal layers were primarily classified as the cluster 1 subpopulation of chief cells.
Atf4 and the unfolded protein response pathway are activated in long-term quiescent chief cells.Subsequently, we compared the transcriptomes of Epcam + and GFP + cells from cluster 1 by using single-cell RNA-seq data to investigate biological signatures associated with the GFP + cells.Gene Ontology (GO) term enrichment analyses indicated that the GFP + cell-associated gene signature included unfolded protein response (Fig. 3a and Supplementary Fig. 3a).Identification of genes shared among the signatures revealed the existence of a complex network of heat shock-related genes whose expression levels were elevated in the GFP + cells (Fig. 3b and Supplementary Fig. 3b).In accordance with these data, single-sample GSEA indicated that the unfolded protein response gene signature was highly expressed in quiescent GFP + cells (Supplementary Fig. 3c).
In addition, computational inference of transcription factor activity by VIPER algorithm 37 revealed that the transcriptional activity of Atf4, a key mediator of the integrated stress response 38,39 , was markedly increased in GFP + cells (Fig. 3c).Accordingly, most Atf4 downstream target genes were upregulated in GFP + cells (Fig. 3d).In addition to Atf4, several other transcription factors involved in the stress response (Hsf1, Atf2, Foxo1) were activated (Fig. 3c).Importantly, VIPER analyses of the ten stomach cell clusters (Fig. 2a, b) indicated that Atf4 was specifically activated in cluster 1 of the chief cells (Fig. 3e).These data indicated that activation of Atf4 and the unfolded protein response were unique hallmarks of the long-term quiescent GFP + chief cells.
Atf4 high quiescent cells comprise a unique subpopulation of chief cells.To examine whether the GFP + cells formed a distinct chief cell subpopulation, we further analyzed single-cell RNA-seq data of the cluster 1 population, in which GFP + cells were highly enriched (Fig. 2d).We stratified the cluster 1 population of chief cells into four subpopulations (1a, 1b, 1c, and 1d) (Fig. 4a, b).Some chief cell markers were ubiquitously expressed in all subpopulations (Pla2g1b, Clps, and Tmed6), while others were preferentially expressed in one of the subpopulations (Chia1 in 1b and Furin in 1d).Of note, a majority of GFP + cells expressed Pla2g1b, but not Chia1 or Troy (Fig. 2e, f), indicating that a fraction of chief cell population constituted GFP + cells.
Notably, the GFP + cells were markedly enriched in the 1a subpopulation (Fig. 4c).In accordance with Atf4 activation and elevated heat shock gene expression in the GFP + cells, elevated expression of the downstream targets of Atf4 (Fig. 4b; Atf3 and Supplementary Fig. 4a) and heat shock genes (Fig. 4b) as well as preferential activation of Atf4 (Fig. 4d) were observed in the 1a subpopulation.In accordance with these findings, immunostaining revealed that most of the GFP + cells near the basal layers expressed Atf4 and heat shock proteins (Hsf1, Hspa8) but not Furin (Fig. 4e).Furthermore, ssGSEA revealed preferential activation of the unfolded protein response in the 1a subpopulation (Supplementary Fig. 4b).These data indicated that the GFP + chief cells were primarily classified into the 1a subpopulation, in which Atf4 and the unfolded protein response were activated.
Subsequently, we examined whether the 1a subpopulation of cluster 1 preferentially expresses chief cell markers.As expected, many of the Atf4 target genes were preferentially expressed in the 1a subpopulation (Fig. 4b, c, Supplementary Fig. 4a), and correlation analyses indicated that the expression of Atf4 target genes (Atf3 and Chac1) was distinct from that of the other known markers (Supplementary Fig. 4d).In accordance with the association of GFP + cells with the 1a subpopulation, Atf3 was uniquely upregulated in GFP + cells among chief cell markers (Supplementary Fig. 4e) and most GFP + cells expressed Atf4 and heat shock proteins (Hsf1, Hspa8) but not Furin (Fig. 4e, f).
In contrast, p57 (Cdkn1c), Gpr30, and Mist1 did not show preferential expression among the subpopulations (Supplementary Fig. 4c).In addition, expression of Lgr5 and Troy was hardly detectable in any of the subpopulations (Supplementary Fig. 4c), which is consistent with the lack of expression of these chief cell markers in cluster 1 (Fig. 2c).These data indicated that the Atf4 + /GFP + cells comprised a distinct quiescent population of chief cells.
External insults initiate the proliferation of Atf4 high quiescent chief cells.Finally, we sought to determine the potential function of GFP + quiescent cells as cellular reserves for tissue restoration following external insults.Rosa26 iH2b-GFP mice were subjected to pulse-chase as in Fig. 1b (chase period: 6 weeks) and then orally administered with indomethacin to determine whether proliferation of the GFP + cells was initiated in response to tissue injury (Fig. 5a).Indomethacin is a commonly used nonsteroidal anti-inflammatory drug that induces gastric lesions and apoptosis of the gastric mucosa 40 .As expected, indomethacin induced extensive destruction of gland structures in the stomach (Fig. 5b).Remarkably, while Mki67 expression was not detected in the GFP + cells without indomethacin treatment, a substantial fraction of the surviving GFP + cells (~20%) expressed Mki67 at 48 hr after treatment (Fig. 5b, c).In addition, a majority of Mki67-expressing GFP + cells expressed Atf4 (Fig. 5d).This demonstrated that the long-term quiescent GFP + /Atf4 + cells were capable of initiating cell proliferation in response to external insults, suggesting that they could function as cellular reserves for damaged tissues.

Discussion
In the present study, we systematically examined the gastrointestinal tract to identify long-term quiescent cells that retained H2b-GFP for extended periods in pulse-chase labeling experiments.We demonstrated that a subpopulation of chief cells in the gastric corpus remained in a slow-cycling state for > 2 months and yet were capable of initiating proliferation following tissue injury.Expression profiles of previously identified cell markers indicate that the identified population was distinct from previously reported reserve cell populations.In addition, the identified subpopulation exhibited a gene signature characterized by the unfolded protein response and activation of Atf4, a transcription factor associated with paligenosis.These results indicate that the identified slow-cycling cells represent a unique subset of chief cells that are associated with elevated stress response and primed for proliferation and tissue regeneration.
After tissue injury, the reserve cells are thought to transform into cycling cell population due to cellular plasticity 8 .The chief cell population, which is located near the basal layer of the corpus gland, is known to harbor reserve cell populations 33 .Burclaff et al. performed BrdU-labeling experiments and showed that chief cells, which rarely proliferate in the absence of damage, undergo massive proliferation after treatment with damageinducing agents 10 .It is likely that the rarely proliferating BrdUlabeled cells that are present in the absence of damage correspond to the H2b-GFP labeled cells in this study.Here, we exploited live cell sorting of GFP-labeled cells to perform a molecular characterization of this rare population of the slow-cycling chief cells.
Previous reports have demonstrated that chief cell populations expressing markers, including Lgr5, Troy, Mist1, Gpr30, and p57, can regenerate the gastric corpus, indicating that cells expressing these markers function as a reservoir to reconstruct damaged tissues 12,13,41 .The expression of these markers was not associated with the GFP + -labeled population in our study, as Mist1, Gpr30, and p57 were detectable at similar levels in four subpopulations of chief cells while Lgr5 and Troy were undetetable in any of them.In contrast, Atf4 activity and the expression of its downstream genes were elevated in the 1a subpopulation.Considering that Atf4 + / GFP + cells preferentially start proliferation after injury, it is possible that Atf4 marks a subpopulation of chief cells that mainly contributes to tissue regeneration after indomethacin treatment.
Atf4 has been reported to be a key mediator of the integrated stress response 38,39 .Interestingly, the identified GFP + cells were also associated with the unfolded protein response, which, together with Atf4, may confer higher resilience to damage-induced stress.Given their predicted anti-stress capacity, the GFP + Atf4 high cells may preferentially survive during external injury.
Notably, induction of ATF3, a transcriptional target of Atf4, is one of the earliest events during the stepwise processes that mediate SPEM cell generation after injury 22 , suggesting that SPEM cells and the GFP + cells share overlapping biological traits.In support of this possibility, another transcriptional target of Atf4 that was upregulated in GFP + cells was Ddit4 (Fig. 3d), which facilitates the initial phase of paligenosis via suppression of mTORC1 42 .In addition, one of the other transcription factors activated in the c1 subpopulation was Sox9 (Fig. 3e), which is another key player during the metaplastic processes of SPEM 21 .Considering the potential association of SPEM with preneoplastic processes 23 , the Atf4 high /GFP + cells may be the source of gastric cancer via transdifferentiation into SPEM-like cells.
Single-cell RNA-seq.Stomach tissues of doxycycline-treated female Rosa26 iH2b-GFP mice were isolated 6 weeks after the beginning of chase periods and subjected to epithelial cell isolation and FACS sorting for Epcam + and GFP + cells.The sorted cells from two female mice were combined and loaded onto a Chromium next GEM chip G (PN-2000177, 10x Genomics) and captured into Gel Beads-in-emulsion (GEMs) by using a Chromium Controller (10x Genomics).cDNA synthesis and sample-indexed library construction were performed using a Chromium next GEM single-cell 3' reagent kit (PN-1000121, 10x Genomics) according to the manufacturer's protocol.Libraries were subjected to sequencing on a HiSeq2500 (Illumina).Mapping of raw sequence reads to mm10 mouse reference genome (version 2020-A, provided by 10x Genomics) and the initial quality control was performed using Cell Ranger software v3.0.2 (10x Genomics).The gene count matrices were imported to Seurat software v4.1.1 running on R v4.1.2.Cell meeting the following criteria were removed from dataset: cells with > 15% of mitochondrial gene counts; cells with >8000 unique featureCounts; cells with <1500 unique featureCounts; cells with positive counts for Ptprc (Cd45) or Pecam1 (Cd31) genes.In total, 4449 cells were subjected to further analyses.
Single-cell RNA-seq data analysis.Analyses of the filtered gene count matrices were performed by Seurat pipeline (https://satijalab.org/seurat/articles/pbmc3k_tutorial.html).The single-cell gene expression counts were log-normalized by using NormalizeData() with a scaling factor of 10,000.The top 2000 most variable genes were identified by FindVariableFeatures(), scaled with ScaleData(), and subjected to principal component analyses to determine top principal components.Subsequently, the top principal components were subjected to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction by RunUMAP(), which was subjected to graph-based cluster detection and visualization using DimPlot.Gene expression levels were represented as violin plots using VlnPlot() or feature plots by using FeaturePlot().Differentially expressed genes (DEGs) for each cluster were identified by Wilcoxon's signed-rank test using FindAllMarkers().A p value (<0.05) and a log2 fold change (log2FC) cutoff (>0.25) were used to select DEGs.Co-expression of quiescent cell markers in the 1a subpopulation was examined by correlation coefficients and hierarchical clustering by Spearman method and ward-D2 method in corrplot (v0.92), respectively.
Enrichment analyses.DEGs (a Bonferroni-adjusted p value < 0.05) between Epcam + and GFP + cells in cluster 1 chief cells were selected by using FindAllMarkers(), and GO enrichment analyses were performed by using clusterProfiler v4.2.2 to identify the top ten most significant GO terms in the biological processes (BP) and molecular functions (MF) categories.Gene concept networks were generated to depict the linkage of genes of selected GO terms by using centplot().ssGSEA for single-cell RNA-seq data was performed using escape v1.5.1.The statistical significances of ssGSEA were calculated using getSignificance().
Quantification of transcription factor activities.The inferred activities of major transcription factors in each cell were determined using VIPER (Virtual Inference of Protein-activity by Enriched Regulon analysis) v1.28.0., which is based on a curated collection of transcription factors and their target genes (DoR-othEA v1.6.0).Transcription factor-target interactions classified as confidence levels A and B were exclusively used as high-quality regulons.VIPER scores were used for split violin plots, feature plots, or heatmaps using pheatmap v1.0.12.
Indomethacin-induced stomach injury.Doxycycline-treated Rosa26 iH2b-GFP mice at 6 weeks after the beginning of chase periods were subjected to fasting overnight with free access to water, and then orally administrated with 20 mg/kg indomethacin in 0.5% carboxymethylcellulose and 20% ethanol.The stomachs of the indomethacin-treated mice after 6 h (n = 4) and 48 h (n = 4) of indomethacin administration were subjected to histological examination and immunofluorescence.Non-treated mice were used as control (n = 4).The numbers of GFP + and GFP/Mki67 + cells within the bottom third of the corpus epithelia were counted to evaluate the emergence of GFP + chief cells initiating proliferation.

Fig. 1
Fig. 1 Stomach epithelia specifically harbor label-retaining long-term quiescent cells.a Genomic structure of the Rosa26 loci after insertion of the inducible H2b-GFP and Tet-on 3 G cassettes.Constitutively expressed Tet-on 3 G activates the TRE3G promoter to express the H2b-GFP gene in a doxycycline (Dox)-dependent manner.The cassette was chemically synthesized with flanking 5' and 3' sequences and inserted in the Rosa26 locus by sgRNA-guided homologous recombination.The open arrowhead indicates the sgRNA targeting site.b Experimental scheme for time course analysis of the H2b-GFP-labeled cells in the digestive tissues.Four-week-old mice were treated with doxycycline for 4 weeks and examined at the indicated time points by immunohistochemistry and/or FACS.c Flow cytometry analysis of the GFP + cells in epithelia of the indicated digestive tissues.d Hematoxylin and eosin (H&E) (upper panel) and immunostaining (lower panel) of the H2b-GFP + cells in the gastric corpus.Black boxes indicate the regions shown in immunostaining.Magnified images of white boxes are shown at bottom column.Scale bar, 100 µm.

Fig. 2
Fig. 2 Long-term quiescent cells in the stomach are localized to the gastric corpus.a Expression profiles of cell type marker genes of the stomach in each cluster (clusters 0-9).6 weeks after the pulse-chase, stomach epithelial cells (Epcam + cells) were subjected to FACS-sorting to isolate GFP + cells.A mixture of Epcam + cells and GFP + cells was used to perform single-cell RNA-seq and classified into ten subpopulations.Gene expression profiles of the markers in each subpopulation are shown in the split violin plots (left: a mixture of Epcam + cells and GFP + cells, right: Epcam + cells).The cellular identity of each cluster was determined based upon expression of Chief cell markers (right panel) and other cell-type specific markers (left panel).b Twodimensional projection of each subpopulation with UMAP (upper panel).The cell type of each subpopulation shown in (a) is summarized (lower panel).c Expression levels of stem cell marker genes in the stomach epithelial cells shown in UMAP Feature plots (upper panels) and split violin plots (lower panels).(left: a mixture of Epcam + cells and GFP + cells, right: Epcam + cells).d A UMAP projection of the GFP + cells (green) and Epcam + cells (gray).The relative distributions of GFP + cells and Epcam + cells are shown in lower panel.The relative ratio of GFP + cell numbers and Epcam + cell numbers in each cluster are represented as fold enrichment values.e H&E and immunostaining of the H2b-GFP + cells in the stomach corpus following a 6-week chase period.Black boxes in the H&E images indicate the regions corresponding to the immunostainings (Marker proteins and GFP).Scale bar, 100 µm.Areas used for merged images (far right panels) are shown as white squares.f Quantification of a fraction of GFP + cells in cells that express the indicated celltype markers.The quantified values are presented as the mean ± SD. n = 3 biologically independent experiments.

Fig. 3
Fig. 3 Atf4 and the unfolded protein response pathway are activated in deeply quiescent chief cells.a Gene Ontology (GO) term enrichment analyses (biological process) of the GFP + chief cells.The p-values and gene counts of top ten GO terms enriched in GFP + cells in comparison to Epcam + cells are shown.The top three terms were shown in blue characters.The p value of each GO term is color-labeled.b A gene concept network for the GO terms enriched in GFP + cells.Blue dots represent the top three terms shown in a (1, protein folding: 2, response to unfolded protein: 3, chaperone cofactordependent protein refolding).Genes that were associated with these terms were shown.Genes belonging to Atf4 regulon (Atf3, Chac1) are shown in red.Extent of induction of these genes in GFP + cells are shown as color shading.c The top ten transcription factors that were differentially activated between GFP + and Epcam + cells.VIPER activation scores of each transcription factor in GFP + and Epcam + cells are color-coded.d Upregulation of the indicated Atf4 regulon genes in GFP + cells relative to Epcam + cells.The Atf4 regulon is composed of 16 genes and the comparison of the regulon genes with detectable expression is shown.Relative expression ratios are shown as log 2 FC values.e VIPER scores of differentially expressed transcription factors in each cluster.The data matrix was sorted by hierarchical clustering (complete linkage method) on both axes and represented as a heatmap.Transcription factors upregulated in the c1 cluster are indicated in red.

Fig. 4
Fig. 4 Atf4 high quiescent cells comprise a unique subpopulation of chief cells.a A UMAP projection of cluster 1 cell classification into four subpopulations.b Split violin plots in each subpopulation of known chief cell marker genes (Mist1, Gif, Pgc, Furin, Pla2g1b, Clps, and Tmed6) and the top 5 heat shock proteins (HSPs) upregulated in the 1a subpopulation (Hsp90aa1, Hspe1, Hspa1a, Dnajb1, and Hspa8) (left: a mixture of Epcam + cells and GFP + cells, right: Epcam + cells).Atf3, a Atf4-regulated gene, is also shown as a marker of the Atf4 high cells.Genes that are preferentially expressed in each subpopulation of cluster 1 are highlighted.c A UMAP projection of the GFP + cells (green) and Epcam + cells (gray) in cluster 1 and relative distributions of GFP + cells and Epcam + cells (right panel).The relative ratios of GFP + cell numbers and Epcam + cell numbers in each cluster is shown as fold enrichment values.d Split violin plot of Atf4 VIPER value of each subpopulation of cluster 1 (left: a mixture of Epcam + cells and GFP + cells, right: Epcam + cells).Feature plot presentation of the Atf4 VIPER value is shown at right panel.e H&E and immunostaining of the H2b-GFP + cells in the stomach corpus at 6 weeks of post-chase periods.Black boxes indicate the regions used for immunostaining with the indicated antibodies.Scale bar, 100 µm.Areas used for merged images (far right panels) are shown as white squares.f Percentage fractions of H2b-GFP + cells expressing the indicated markers shown in e.The GFP + cells located within bottom third of the corpus are evaluated and presented as the mean ± SD. n = 3 biologically independent experiments.

Fig. 5
Fig. 5 Atf4 high quiescent chief cells are capable of initiating proliferation in response to external insults.a Experimental scheme for Indomethacin (IND)induced stomach damage models.Indomethacin was administered orally at 6 weeks after the start of post-chase periods and histological analyses was performed at 6 h or 48 h after IND treatment.b H&E and immunostaining of the H2b-GFP + cells in the stomach corpus following indomethacin-induced injury.At 48 hr after indomethacin treatment, Mki67 expression was unevenly distributed in GFP + cells probably due to variegated effects of the treatment, and representative images near the basal layers where GFP + cells highly express Mki67 are shown.Magnified images of white boxes are shown at bottom column.Arrowheads indicate Mki67 + /GFP + cells.-IND: untreated control.Scale bar, 100 µm.c Average fraction of Mki67 + cells among GFP-positive and negative chief cells at the indicated time points after the IND-induced injury.Mki67 staining was evaluated in GFP + / Pla2g1b + cells and GFP -/ Pla2g1b + cells.The quantifed values are presented as the mean ± SD. n.s., not significant.n = 4 biologically independent experiments.d Representative images of triple staining of Mki67, GFP, and Atf4 at the indicated time after indomethacin treatment are shown.Merged images of white boxes are shown at far right.Arrowheads indicate Mki67 + /GFP + /Atf4 + cells.-IND: untreated control.Scale bar, 100 µm.
Statistics and reproducibility.Data are represented as mean ± standard deviation.For calculation of p values, two-tailed Student's t test was performed except comparison of data shown in Supplementary Fig. 4e, for which the Wilcoxon test was used for the calculation.The number of replicates was described in figure legends.